The present invention relates to improved syntheses of known and novel benzothiazine dioxide which are potent and selective endothelin antagonists. The processes of the instant invention are improved over those recited in U.S. Pat. No. 5,599,811 which is hereby incorporated by reference.
The compounds of the instant invention exhibit very significant improvements over those described in U.S. Pat. No. 5,599,811. These improvements include: binding affinity to the ETA receptor, ETA selectivity, functional activity, long pharmacokinetic half-life, high bioavailability, in vivo activity in inhibiting the pressor effect caused by bET-1, oral activity with relatively long duration of action, and efficacy in acute hypoxic pulmonary hypertension in rats.
The processes of the instant invention provide more facile syntheses with higher yields. They are short, clean, reproducible and no tedious chromatography is needed. Moreover, the processes are scaleable and therefore useful for large-scale development.
The present invention also relates to antagonists of endothelin useful as pharmaceutical agents, to pharmaceutical compositions which include these compounds and a pharmaceutically acceptable carrier, and to pharmaceutical methods of treatment. More particularly, the compounds of the present invention are antagonists of endothelin useful in treating elevated levels of endothelin, acute and chronic renal failure, essential renovascular malignant and pulmonary hypertension, cerebral infarction and cerebral ischemia, cerebral vasospasm, cirrhosis, septic shock, congestive heart failure, endotoxic shock, subarachnoid hemorrhage, arrhythmias, asthma, preeclampsia, atherosclerotic disorders including Raynaud""s disease and restenosis, angina, cancer, benign prostatic hyperplasia, ischemic disease, gastric mucosal damage, hemorrhagic shock, ischemic bowel disease, and diabetes.
Also, the compounds will be useful in cerebral ischemia or cerebral infarction resulting from a range of conditions such as thromboembolic or hemorrhagic stroke, cerebral vasospasm, head injury, hypoglycemia, cardiac arrest, status epilepticus, perinatal asphyxia, anoxia such as from drowning, pulmonary surgery, and cerebral trauma.
This invention is improved processes for the preparation of compounds of Formula I 
or a pharmaceutically acceptable salt thereof wherein
R1 is hydrogen, alkyl, or alkoxy;
R2 is hydrogen or alkoxy;
R3 is alkyl or alkoxy;
R2 and R3 may be joined to form a ring 
R4 is hydrogen or alkyl;
R5 is hydrogen, alkyl, alkoxy, halogen at the 2 or 3, or 4, or 5 positions or R5 is a 3,4-methylenedioxo; and
R6 is CF3, halogen, alkyl, benzyl, phenyl, hydroxy, or pyrrole comprising:
a) alkylating a compound of formula A 
xe2x80x83using sodium hydride in DMF followed by reaction with methyl bromoacetate to produce a compound of formula B 
b) combining compound B in THF with a solution of TiCl4 in solvent at xe2x88x9278xc2x0 C., treating with triethylamine and quenching with an acid to produce a compound of formula C 
c) treating compound C with triflic anhydride in a solvent in the presence of pyridine for from 1 to 5 hours to produce a compound of formula D 
d) coupling the compound D with a boronic acid of formula X 
xe2x80x83in DMF and toluene in the presence of a palladium catalyst and potassium carbonate at about 100xc2x0 C. to produce a compound of Formula 1.
The free acid of Formula 1 is obtained by sponification of the ester with, for example, LiOH in THF/MeOH or in dioxane. Any strongly alkaline solution in methanol can be used.
Compounds of the invention are those prepared by the above process, especially those selected from:
4-(3,5-Dimethoxy-phenyl)-2-(2-trifluoromethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Chloro-phenyl)-4-(3,5-dimethoxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Bromo-phenyl)-4-(3,5-dimethoxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Chloro-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2-Benzyl-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
2-(2,6-Dimethyl-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid monosodium salt;
4-(7-Methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-ethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-propyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-isopropyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-butyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-fluoro-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-hydroxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2,3-dichloro-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2,4-dichloro-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2-chloro-4-methoxy-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(3-chloro-2-methyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(2,6-dimethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-1,1-dioxo-2-(2-pyrrol-1-yl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-Benzo[1,3]dioxol-5-yl-2-(3,4-dimethyl-isoxazol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid;
4-(6-Methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid; and
4-(3,5-Dimethyl-phenyl)-2-(2-trifluoromethyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid.
The invention is also a process for the preparation of a compound of Formula 1
comprising:
a) treating aryl bromide with n-butyl lithium followed by zinc bromide to generate an aryl zinc bromide of Formula Y 
b) reacting the product of step a) above with 
xe2x80x83in THF in the presence of a palladium catalyst to produce a compound of Formula 1
The free acid of Formula 1 is obtained by sponification of the ester.
The following three compounds are obtained by the above process:
2-(2-Bromo-phenyl)-4-(7-methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid monosodium salt;
4-(7-Ethyl-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid, potassium salt; and
4-Benzo[1,3]dioxol-5-yl-2-(2-benzyl-phenyl)-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid.
The invention is also a process for the preparation of a compound of formula B 
comprising:
a) reacting a phenyl sulfonyl chloride with 1 equivalent of aniline to produce a sulfonamide of formula F 
b) lithiating the product of step a) above at low temperatures and quenching with CO2 to form a compound of formula G 
c) treating the product of step b) above with acetic anhydride and a catalytic amount of methanesulfonic acid to produce a compound of formula H 
d) treating the product of step c) above with NaOMe followed by methyl bromoacetate to produce a compound B as above.
Compound B is carried over to Formula 1 as described before.
Compounds of the invention are those prepared from the above process, especially 4-(7-Ethyl-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihiydro-1xcex6benzo[e][1,2]tiazine-3-carboxylic acid methyl ester.
The sodium and potassium salts of the above compound are preferred.
The invention is also a process for the preparation of a compound of Formula 1 which comprises
a) refluxing 
xe2x80x83with one equivalent of pyridine and DMAP to produce cyclic intermediate of formula H 
b) treating the product of Step a) above with potassium hexamethyldisilylazide followed by t-butyl acetate in THF to produce the keto ester of formula I 
c) brominating the product from Step b) above to produce an intermediate of formula J 
d) cyclizing the product of Step c) above using potassium carbonate in DMF to produce enol of formula K 
The enol is then used to produce a compound of Formula 1 above.
Compounds of the invention are those prepared by the above process and especially those selected from:
2-(6-Methyl-benzo[1,3]dioxol-5-yl)-4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester;
2-(2-Bromo-phenyl)-4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester;
2-(2-Chloro-phenyl)-4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester; and
2-(6-Chloro-benzo[1,3]dioxol-5-yl)-4-hydroxy-1,1-dioxo-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid tert-butyl ester.
The invention is also a process for the preparation of a compound of Formula 1 comprising:
a) reacting 2-methylanaline with benzenesulfonyl chloride to produce a compound of formula F 
b) treating the product of step a) above with n-butyl lithium and then with 3-methoxy-4,5-methylenedioxybenzaldehyde to produce a compound of formula M 
c) treating the product of step b) above with sodium hydride followed by methyl bromoacetate to produce a compound of formula N 
d) oxidizing the product of step c) above to produce the corresponding keto ester of formula O 
e) cyclizing the product of step d) above by treating with a base or a Lewis acid in an appropriate solvent to produce the corresponding ester of Formula 1
Compounds of the invention are those prepared by the above process, especially 4-(7-Methoxy-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-o-tolyl-1,2-dihydro-1xcex6-benzo[e][1,2]thiazine-3-carboxylic acid methyl ester.
The invention is also a pharmaceutical composition of the compounds of Formula 1 above comprising a therapeutically effective amount of a compound of Formula 1 in admixture with a pharmaceutically acceptable carrier.
The compounds of the invention are useful in inhibiting elevated levels of endothelin comprising administering to a host in need thereof a therapeutically effective amount of a compound of Formula I in unit dosage form. They are also useful in treating subarachnoid hemorrhage, essential, renovascular, malignant and pulmonary hypertension, congestive heart failure, cerebral ischemia, or cerebral infarction.
In the compounds of Formula I, the term xe2x80x9calkylxe2x80x9d means a straight or branched hydrocarbon radical having from 1 to 12 carbon atoms unless otherwise specified and includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, allyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, and dodecyl.
xe2x80x9cHalogenxe2x80x9d is fluorine, chlorine, bromine or iodine.
The term xe2x80x9calkoxyxe2x80x9d is an alkyl group as described above attached by oxygen to the rest of the molecule. Preferred alkoxy group are from 1 to 4 carbon atoms.
The host receiving a compound of the invention is a mammal, particularly a human.
The compounds of Formula 1 above may be prepared by several methods. These methods are illustrated in Schemes 1 through 3 and in a detailed manner by way of illustration in the example section of the specification.
Scheme 1 illustrates the general procedure for the preparation of the compounds of Formula 1. Synthesis of the intermediate A was described in U.S. Pat. No. 5,599,811. Reaction of methyl-2-(chlorosulfonyl)benzoate with an aniline in methylene chloride with 1 to 2 equivalents of pyridine and a catalytic amount of DMAP at room temperature overnight affords the sulfonamide A. The reaction can also be run using neat pyridine as the solvent. Alkylation of A was achieved by treatment of A with sodium hydride in DMF followed by reaction with methyl bromoacetate to give intermediate B. B can be converted to the key intermediate C via Claisen cyclization which were found to be promoted by at least two different catalysts. In condition I, the intermediate B in methylene chloride was added to a solution of 2 equivalents of TiCl4 in methylene chloride at xe2x88x9278xc2x0 C. followed by the treatment with 2.2 equivalents of triethyl amine. The reaction was quenched with 1N HCl followed by aqueous work up and recrystallization. The condition II is similar to condition I with the use of 1.2 equivalents of TiCl2(OTf)2 (Bull. Chem. Soc. Jpn, 1989;62:1917) followed by quench with pH 7 phosphate buffer and extractive workup. The cyclized enol C was treated with triflic anhydride in solvents such as methylene chloride in the presence of 2 to 5 equivalents of pyridine for 1 to 5 hours to afford the triflate D quantitatively. The coupling reaction was carried out between the triflate D and the corresponding boronic acid in DMF and toluene (ratio of 1:4) in the presence of 10 mole % tetrakis(triphenyl phosphine)palladium as the catalyst and 1 to 2 equivalents of potassium carbonate under reflux for 2 hours. The reaction mixture was filtered, and the product was purified by column chromatography or by recrystallization. The free acid of the Formula 1 was obtained by sponification of the ester with aqueous LiOH in THF/MeOH or in dioxane. The free acid can be converted, for example, to a sodium or potassium salts, which are much more soluble in H2O, by treating it with one equivalent of NaOH or KOH in MeOH followed by recrystallization.
Scheme 1a indicates an alternative assembly reaction for the formation of the 4-aryl benzothiazine derivatives exemplified by intermediate E. The coupling reaction was conducted by reacting an aryl zinc bromide, prepared by treating an aryl bromide with one equivalent of n-butyl lithium in THF followed by zinc bromide, with the intermediate D in the presence of 10% tetrakis(triphenylphosphine)palladium as the catalyst under reflux for 2 hours.
The Scheme 1 was further modified to replace a relatively expensive starting material, methyl-2-(chlorosulfonyl)benzoate, with phenyl sulfonyl chloride. The process (Scheme 1b) is proven to be more economical and workable in large scale. The sulfonamide F was formed by reacting the phenyl sulfonyl chloride with one equivalent of aniline under similar condition as described for A or using an equal amount of saturated sodium bicarbonate solution in THF. It was lithiated with n-BuLi at low temperature and quenched with CO2 to form the intermediate G in 92% yield. The acid G was then treated with acetic anhydride with a catalytic amount of methanesulfonic acid to form the intermediate H. This cyclic compound was treated with NaOMe followed by quenching with methyl bromoacetate to form B directly in 71% yield. 
Several compounds in the Examples were synthesized through a synthesis outlined in Scheme 2. Compound A, obtained as described in Scheme 1 was refluxed in xylene for 16 hours with 1 equivalent of pyridine and a catalytic amount of DMAP to afford the cyclic intermediate H. This compound was treated with potassium hexamethyldisilylazide followed by t-butyl acetate in THF at xe2x88x9278xc2x0 C. to yield the keto ester I. Bromination of I was carried out with NBS in carbon tetrachloride to obtain J which is in turn cyclized via treatment with potassium carbonate in DMF. The enol K was converted to the ester L with similar procedures illustrated in Scheme 1. Finally, the t-butyl ester was removed by treatment with TFA in methylene chloride at room temperature to give the free acid. 
Scheme 3 illustrates an alternative synthesis that has been successfully utilized in the synthesis of certain analogs. This is illustrated by the synthesis of Example 8. Intermediate F was treated with 2 equivalents of n-butyl lithium at xe2x88x9278xc2x0 C. to generate the dianion. Then a solution of 3-methoxy-4,5-methylenedioxybenzaldehyde in tetrahydrofuran was added to the reaction mixture. It was then warmed to 0xc2x0 C. over 1.5 hours. Aqueous work up afforded M which was treated with sodium hydride followed by methyl bromoacetate to give N. Jones oxidation of N was carried out in acetone to afford the keto ester O in 60% yield. The cyclization of O can be promoted by a base or a Lewis acid such as titanium bis-chloro-bis-triflate in an appropriate solvent such as methylene chloride. The ester E was purified by column chromatography to provide pure sample in 53% yield. The esters synthesized through this route were sponified according to procedures described in Scheme 1. 
Some of the compounds of Formula 1 are capable of further forming both pharmaceutically acceptable base salts. All of these forms are within the scope of the present invention.
Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,Nxe2x80x2-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge S M, et al., xe2x80x9cPharmaceutical Salts,xe2x80x9d Journal of Pharmaceutical Science, 1977;66:1-19).
The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
Certain of the compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention.
Certain of the compounds of the present invention possess one or more chiral centers and each center may exist in the R or S configuration. The present invention includes all enantiomeric and epimeric forms as well as the appropriate diastereomeric mixtures thereof.
Test Protocols:
The compounds of Formula 1 are valuable antagonists of endothelin. The tests employed indicate that compounds of the invention possess endothelin antagonist activity. Thus, the compounds were tested for their ability to inhibit [125I]-ET-1([125I]Endothelin-1) binding in a receptor assay. Selected compounds were also tested for antagonist activity by inhibition of ET-1 stimulated arachidonic acid release and ET-1 stimulated vasoconstriction. The following testing procedures were used (Doherty A M, et al., xe2x80x9cDesign of C-Terminal Peptide Antagonists of Endothelin: Structure-Activity Relationships of ET-1 [16-21, D-His16]xe2x80x9d, Bioorganic and Medicinal Chemistry Letters, 1993;3:497-502).
The following cultured cells were used in binding experiments: Ltk-cells expressing recombinant human ETAR (HETA), and CHO-K1 cells expressing recombinant human ETBR (HETB).
Membranes were prepared from cultured cells by lysing cells in cold lysis buffer (5 mM HEPES, 2 mM EDTA, pH 7.4) and homogenizing with a Dounce xe2x80x9cAxe2x80x9d homogenizer. The homogenate was centrifuged at 30,000xc3x97g for 20 minutes at 40xc2x0 C. Membrane pellets were suspended in cold buffer containing 20 mM Tris, 2 mM EDTA, 200 xcexcM Pefabloc, 10 xcexcM phosphoramidon, 10 xcexcM leupeptin, 1 xcexcM pepstatin at pH 7.4 and frozen at xe2x88x9280xc2x0 C. until use. Membranes were thawed and homogenized with a Brinkmann Polytron then diluted in tissue buffer containing 20 mM Tris, 2 mM EDTA, 200 xcexcM Pefabloc, and 100 xcexcM bacitracin (pH 7.4). Radioligand and competing ligands were prepared in binding buffer containing 20 mM Tris, 2 mM EDTA, and 0.1% BSA.
Competing binding assays were initiated by combining membranes, [125I]-ET-1 (40 pM) and the competing ligand in a final volume of 250 xcexcL and incubating for 2 hours at 37xc2x0 C. The assay was terminated by filtration over Whatman GF/B filters which were presoaked with 50 mM Tris, pH 7.4 containing 0.2% BSA and 100 xcexcM bacitracin. Nonspecific binding was defined as binding in the presence of 100 nM ET-1.
Antagonist activity is measured by the ability of added compounds to reduce endothelin-stimulated arachidonic acid release in cultured vascular smooth muscle cells. [3H]Arachidonic Acid Loading Media (LM) is DME/F12+0.5% FCSxc3x970.25 mCi/mL [3H] arachidonic acid (Amersham). Confluent monolayers of cultured rabbit renal artery vascular smooth muscle cells were incubated in 0.5 mL of the LM over 18 hours, at 37xc2x0 C., in 5% CO2. The LM was aspirated and the cells were washed once with the assay buffer (Hank""s BSS+10 mM HEPES+fatty acid-free BSA (1 mg/mL)), and incubated for 5 minutes with 1 mL of the prewarmed assay buffer. This solution was aspirated, followed by an additional 1 mL of prewarmed assay buffer, and further incubated for another 5 minutes. A final 5-minute incubation was carried out in a similar manner. The same procedure was repeated with the inclusion of 10 xcexcL of the test compound (1 nM to 1 xcexcM) and 10 xcexcL ET-1 (0.3 nM) and the incubation was extended for 30 minutes. This solution was then collected, 10 mL of scintillation cocktail was added, and the amount of [3H] arachidonic acid was determined in a liquid scintillation counter.
Male New Zealand rabbits were killed by cervical dislocation and exsanguination. Femoral and pulmonary arteries were isolated, cleaned of connective tissue, and cut into 4-mm rings. The endothelium was denuded by placing the rings over hypodermic tubing (32 gauge for femoral rings and 28 gauge for pulmonary rings, Small Parts, Inc., Miami, Fla.) and gently rolling them. Denuded rings were mounted in 20 mL organ baths containing Krebs-bicarbonate buffer (composition in mM: NaCl, 118.2; NaHCO3, 24.8; KCl, 4.6; MgSO4 7H2O, 1.2; KH2PO4, 1.2; CaCl2 2H2O; Caxe2x80x94Na2 EDTA, 0.026; dextrose, 10.0), that was maintained at 37xc2x0 C. and gassed continuously with 5% CO2 in oxygen (pH 7.4). Resting tension was adjusted to 3.0 g for femoral and 4.0 g pulmonary arteries; the rings were left for 90 minutes to equilibrate. Vascular rings were tested for lack of functional endothelium (ie, lack of an endothelium-dependent relaxation response to carbachol (1.0 nM) in norepinephrine (0.03 nM) contracted rings. Agonist peptides, ET-1 (femoral), and S6c (pulmonary), were cumulatively added at 10-minute intervals. The ET antagonists were added 30 minutes prior to adding the agonist.
Inhibition of bET-1 Induced Pressor Effect in Rat
Male Sprague-Dawley (Charles River Laboratories, Kingston, Ontario, Canada) weighing 250 to 350 g are anesthetized (Inactin, 120 mg/kg, I.P.) and acutely instrumented with a carotid artery catheter to monitor arterial blood pressure and with a jugular vein catheters to administer intravenous drugs. Once instrumented, the rats are ganglionic-blocked with mecamylamine (1.25 mg/kg, I.V.) to prevent hemodynamic reflexes and then challenged with big endothelin-1 (bET-1) (1.0 nmol/kg, I.V.). The peak arterial pressor response in rats pre-treated with selected compounds of invention compared to vehicle-treated rats is used to determine activity expressed as % inhibition. For I.V. activity selected compounds of invention was dose 10 minutes before the bET-1 challenge, and for oral activity selected compounds of invention was administered via oral gavage 8 or 24 hours before the bET-1 challenge.
For example, Example 10 is a potent inhibitor (IC50=0.6 nM) of ET-1 binding to human ETA receptors. It also has potent fumctional antagonist activity in human pulmonary artery smooth muscle cells blocking ET-1 induced Ca++ transients via the ETA receptor (IC50=0.2 nM). It exhibits antagonism to ET-1 stimulated vasoconstriction in rabbit femoral artery with pA2 value of 7.7. In vivo, ETA blockade with oral Example 10 was demonstrated through inhibition of bET-1 pressor response in the rat (10 mg/kg produced 44% inhibition at 8 hours postdose and 30 mg/kg produced a 48% inhibition at 24 hours postdose) and dog (15 mg/kg produced 25% inhibition at 24 hours postdose).
Oral Example 10 inhibited acute hypoxic pulmonary hypertension in the rat in a dose dependent manner with the ED50=0.8 mg/kg (the plasma EC50=0.046 xcexcg/mL). Example 10 was well absorbed orally both in rat and dog. Terminal Ielimination txc2xd""s were determined to be 8.5 and 2.2 hours in rat and dog, respectively, and bioavailability is 77% and 100% in rat and dog.
The compounds of the present invention can be prepared and administered in a wide variety of oral and parenteral dosage forms. Thus, the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compounds of the present invention can be administered by inhalation, for example, intranasally. Additionally, the compounds of the present invention can be administered transdermally. It will be obvious to those skilled in the art that the following dosage forms may comprise as the active component, either a compound of Formula 1 or a corresponding pharmaceutically acceptable salt of a compound of Formula 1.
For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.
In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term xe2x80x9cpreparationxe2x80x9d is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions. For parenteral injection liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired.
Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, table, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
The quantity of active component in a unit dose preparation may be varied or adjusted from 0.1 mg to 100 mg preferably 0.5 mg to 100 mg according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.
In therapeutic use as antagonists of endothelin, the compounds utilized in the pharmaceutical method of this invention are administered at the initial dosage of about 0.01 mg to about 100 mg/kg daily. A daily dose range of about 0.01 mg to about 10 mg/kg is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.